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Notes: To identify proteins that may initiate the formation of β2-microglobulin (β2-m) amyloid fibrils in dialysis related amyloidoisis (DRA), the authors screened a human synovial cDNA library using β2-m as bait in a yeast two-hybrid system. Procollagen C-proteinase enhancer-1 (PCPE-1) was shown to interact with β2-m. This interaction was confirmed by immunoprecipitation, solid-phase binding and pull-down assays. Full-length PCPE-1 cDNA was cloned into the pTNT™ Vector and expressed using a TNT® Transcription/Translation System. Pull-down assays were performed by immobilizing the GST-β2-m fusion protein using the MagneGST Ni Particles, adding the in vitro translated PCPE-1 protein, washing the particles, then performing SDS-PAGE and Western blotting using anti-PCPE-1 antibodies. (3966)

Notes: The authors examined the interaction between various proteins involved in mRNA deadenylation and degradation using GST pull-down assays. Cytoplasmic poly(A)-binding protein (PABPC1) was expressed in E. coli as a GST fusion and immobilized using the MagneGST™ Glutathione Particles. This bait protein was incubated with various [35S]Methionine-labeled prey proteins expressed in the TNT® Coupled Reticulocyte Lysate System. Bait:prey complexes were washed, eluted with 1X SDS loading buffer, then analyzed by SDS-PAGE to determine which proteins interacted. (3782)

Notes: The authors characterized a juvenile hormone response element in Drosophila melanogaster (DmJHRE1) and identified two proteins that bound to a DmJHRE1 affinity column. Proteins eluted from the column were digested with Sequencing Grade Modified Trypsin, subjected to liquid chromatography-tandem mass spectrometry and identified as FKBP39 and Chd64. DmJHRE1 transcription regulatory activity was confirmed using reporter constructs with DmJHRE1 sequences regulating expression of firefly luciferase in Drosophila L57 and S2 cells. A vector with Renilla luciferase and the Autographa californica multicapsid nucleopolyhedrovirus IE1 promoter was used for normalization. Luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. Potential interactions between FKBP39, Chd64 and several candidates proteins for the JH receptor were examined using the MagneGST™ Pull-Down System. Each bait protein was expressed as a GST-fusion protein in E. coli and immobilized using MagneGST™ Glutathione Particles. [35S]Methionine-labeled prey proteins were expressed using the TNT® T7 Quick Coupled Transcription/Translation System. (3784)

Notes: Sex-determining Y-box (SOX) 6 is a transcription factor downregulated in obesity-related insulin-resistant animals. The authors examined the interaction between SOX 6 and β-catenin, a protein that modulates cyclin D1 promoter activity. To characterize the physical interaction, in vitro binding assays were performed using GST-fused SOX 6 and deletion mutants of β-catenin, which were expressed as 35S-labeled proteins in the TNT® T7 Quick Coupled Transcription/Translation System. The GST-fusion proteins were bound to MagneGST® particles and allowed to interact with the β-catenin mutants. Purified GST was used as a negative control to determine nonspecific protein binding. The authors were able to identify the protein domains necessary for SOX 6/β-catenin interaction. Similar binding assays were performed with GST-β-catenin and 35S-labeled T-cell factor in the presence or absence of SOX 6 to show that SOX 6 does not interfere with the binding of β-catenin to TCF. (3685)

Notes: To better understand the specificity and function of the 12 amino acid PAM2 (PABP-interacting motif 2) domain from the HECT E3 ubiquitin ligase (HYD), possible binding partners were identified. One set of potential partners, the anti-proliferative Tob proteins, were radiolabeled with 35S and expressed in vitro using a TNT® Quick Coupled Transcription/Translation reaction. The PAM2 domain from HYD and poly(A) binding protein were fused with a GST domain, expressed and purified from E. coli. Using the MagneGST™ Pull-Down System, the interaction between the PAM2 domain and the Tob proteins was confirmed by autoradiography. (3400)

Notes: These authors characterized the interaction between whirlin, a protein component of stereocilia in the inner ear, and the membrane-associated guanylate kinase protein p55. Portions of p55 and whirlin were expressed in the TNT® T7 Coupled Transcription/Translation System as 35S-labeled protein. GST-fusion proteins of the guanylate kinase of p55, the PDZ3 domain of whirlin or GST alone were expressed in E. coli. Interaction between the 35S-labeled proteins and the GST-fusion proteins were examined in GST pull-down assays using the MagneGST® Pull-Down System. GST alone was used as a negative control. Briefly, the MagneGST® particles were washed eight times with wash/binding buffer supplemented with 0.05% Nonidet® P-40, and bound proteins were resuspended in 2X SDS sample buffer and analyzed on 12.5% SDS polyacrylamide gels. (3683)

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